| [Objective]Pathologic myopia (PM) is one of the most serious eye disease to blindness in the clinic, especially for adults. There is no clinical successful therapeutic intervention that benefit at present. Studies on pathogenesis had shown that various agents had taken sclera as the essential target structure, and the weakness of sclera might be either the result of PM, or of the risk factor that could induce related lesions. Therefore, enhancement of sclera biomechanical strength could be as a referenced method to alleviate of the development of PM.The purpose of this study is to observe the effects of glyceraldehyde cross-linking on sclera biomechanical strength and experimental myopia, based on the model of the guinea pig form deprivation myopia (FDM). New therapeutic strategy for PM was explored from the view of scleral remodeling control.[Materials and Methods]1In vitro experiment:15three-color guinea pigs aged three weeks were randomly divided into3groups. Each group had5guinea pigs. The right eyes were treated and the left eyes were used as untreated controls.2mm x6mm sclera strip was taken after the eyeball extraction and treated respectively with glyceraldehyde at concentration of0.005mol/L,0.05mol/L, and foundational solution without glyceraldhyde at room temperature. After4-day cross-linking, biomechanical stress-strain measurements of all scleral strips were performed using a microcomputer-controlled biomaterial testing device. The parameters ultimate stress (σmax)(MPa) and ultimate strain (εmax)(%) and6%elastic modulus (MPa) were used for analysis.2In the MDF group, latex balloons were modified into facemasks, which only covered the right eye of the animals, leaving the left eye, nose, mouth and ears exposed. Guinea pigs breeding at room temperature, normal circadian rhythms Ocular axial length, vitreous cavity length and refractive error were measured before form deprivation and at mask day7,14,21.3Posterior scleral cross-linking:35three weeks aged guinea pigs were randomly divided into5groups:A, B, C, D, E. Each group had7guinea pigs. The right eye was set as the experimental eye, the left eye was used as control. Group A:7days mask; Group B:21days mask, plus physiological saline retrobulbar injection at mask day1,8,15; Group C:21days mask, plus0.05mol/L glyceraldehyde retrobulbar injection at mask day1,8,15; Group D:21days mask, plus0.5mol/L glyceraldehyde retrobulbar injection at mask day1,8,15; Group E:normal control group. Ocular axial length, vitreous cavity length and refractive error were measured before form deprivation and at mask day7,14,21.2mm x6mm sclera strip was taken after the eyeball extraction at the end of experiment, biomechanical stress-strain measurements of all scleral strips were performed using a microcomputer-controlled biomaterial testing device. The parameters ultimate stress (amax)(MPa) and ultimate strain (εmax)(%) and6%elastic modulus (MPa) were used for analysis.4Ultrastructure examination:15three weeks aged guinea pigs were randomly divided into5groups. The right eye was set as the experimental eye, the left eye was used as control. Administration of posterior scleral cross-linking was performed referring to protocols described in step3. Animals were humanly killed at the end of the experiment. Ocular ultrastructure structures examination of sclera, choroid, retina was performed by hematoxylin-eosin staining. The expression of MMP-2was detected by immunohistochemical staining. The effects of glyceraldehyde on adjacent tissues, including limbus, the extraocular muscles and optic nerve were also detected.[Results]1. The stress-strain curves showed the prominent increase of biomechanical stiffness in vitro. After the cross-linking treatment, the ultimate stress, the ultimate strain,6%elastic modulus were (7.198±0.991)MPa,(21.480±0.853)%and (22.808±2.159) MPa in the controls.6%elastic modulus of0.005mol/L. glyceraldehyde group and0.05mol/L glyceraldehyde group were (25.903±0.892)MPa(P=0.019)and(36.156±2.026)MPa(P=0.000), increased13.57%and58.52%versus the control group respectively. The ultimate stress of0.005mol/L glyceraldehyde group and0.05mol/L glyceraldehyde group were (10.386±1.023) MPa (P=0.0012) and (14.851±1.602) MPa (P=0.000) increased44.30%and106.32%respectively. The ultimate strain of groups0.005mol/L, glyceraldehyde group and0.05mol/L glyceraldehyde group were (19.320±0.672)%(P=0.002) and (14.140±1.001)%(P=0.000), decreased10.06%and34.17%respectively.2. Form deprivation, at the end of the experiment, the experimental eyes appeared the increase of the the vitreous cavity length, the axial length and myopia. In group A, group B and group C. the length of the vitreous cavity, the axial length and refractive error, compared with the fellow eye the difference was statistically significant statistically (Pvitreous cavity=0.018.0.002.0.001; Paxial=0.019.0.002.0.000; Prefraction=0.000,0.005,0.001). In group D and group E, the length of the vitreous chamber, axial length compared with the fellow eye, the difference was not statistically significant (P vitreous cavity=0.607,0.539; Paxial=0.607.0.539). The experimental eye and the fellow eye of group D, the difference of diopter was statistically significant (Prefraction=0.020), but diopter of group E was no significant difference.(Prefraction=0.580).3. At the end of the experiment, diopter change in value of experimental eye of group B, C, D, E was significantly different, the overall difference was statistically significant (F=61.249, P=0.000). Diopter change value of group B,C,D versus the normal control group, the difference was statistically significant (PB=0.000. Pc=0.000, PD=0.000):but the difference of axial length changes in the value was no statistical significance (PB=0.430, Pc=0.840. PD=0.386).4. At the end of the experiment, B and E groups contralateral eye, the added value of group B of the vitreous cavity length compared with group E, the difference was statistically significant (t=-3.689, P=0.002); the difference of the increase in axial length was not statistically significant (t=-1.295, P=0.216). 5. Axial length and refractive error of experimental eye of group B was highly negative correlated (r=-0.832, P=0.000); vitreous cavity length and refractive error of experimental eye of group B was highly negative correlated (r=-0.804, P=0.000);the axial length and refraction of right eye of group E was a moderate negative correlation (r=-0.604, P=0.000); axial length of group E eyes was highly correlated (r=0.940, P=0.000).6. At the end of the experiment, the ultimate stress and6%elastic modulus of group B experimental eye was (7.988±3.677) MPa (P=0.002) and (19.938±4.871) MPa(P=0.001), decreased10.06%and34.17%versus the fellow eye respectively, the Ultimate strain was (28.6±3.6)%(P=0.034) increased19.17%respectively. After the cross-linking treatment, the ultimate stress and6%elastic modulus of group C experimental eye was (9.244±0.806)MPa(P=0.001)and(26.180±4.388) MPa (P=0.031). decreased23.13%and13.34%respectively. the ultimate strain was (26.2±1.0)%(P=0.016).increased12.93%respectively. The ultimate stress of group D experimental eye was (12.476±2.507) MPa (P=0.580).decreased5.50%,6%elastic modulus was (30.446±3.410) MPa (P=0.314) increased6.53%, ultimate strain was (23.8±1.8)%(P=0.253). decreased4.42%respectively. The ultimate stress of group D experimental eye increased35.85%versus group B,6%elastic modulus increased52.70%, ultimate strain decreased16.78%. The stress-strain curves showed the prominent increase of biomechanical stiffness.7. Ultrastructure examination:As the development of guinea pig FDM, Ultrastructure examination showed that, decreased scleral thickness with fibers lined up in order, decreased choroidal thickness with vascular lumen, lack of sinus cavity. The retinal structure was normal. Expressions of MMP-2were mainly in the episcleral tissue, the stroma of choroid, and the outer plexiform layer. After glyceraldehyde cross-linking, the loose episcleral tissue became dense and structured, and the adhesion with sclera became tight. Scleral thickness decreased while the dense of sclera cells increased. Lumens and cavities in the choroid became obvious, and no significant thickness change was noticed, The retinal structure was morphologically normal with distinct structural layers and regular arrangement of retinal photoreceptor cells, without inflammatory cells Infiltration. Expressions of MMP-2were mainly in the episcleral tissue. MMP-2in the stroma of choroid, and the outer plexiform layer.[Conclusion]1. Glyceraldehyde is a safe and effective cross-linking agent that could significantly enhance the sclera biomechanical strength.2. The monocularly-deprived facemask could successfully induce guinea pig for animal myopia model, which is axial extension myopia, especially the vitreous cavity length.3. With increasing of the axial length and progressing of myopia, the scleral biomechanical strength is significantly reduced.4. The correlation between MMP-2and progression of myopia model is meaningful, and glyceraldehyde could reduce the positive expression of MMP-2.5. Glyceraldehyde cross-linking method could effectively control the development of pathologic myopia in animal model. New therapeutic strategy for PM was explored from the view of scleral remodeling control. |
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